Listeria monocytogenes is the etiologic agent of the life-threatening foodborne illness listeriosis and a model organism for the study of intracellular parasitism. Two major mechanisms of host cell invasion by intracellular pathogens have been described. Pathogens express surface invasins that specifically activate host cell receptors, or produce a secretion apparatus that injects effectors into the host cell cytosol, leading to the activation of the host cell endocytic machineries that internalize the pathogen. We discovered a third mechanism of invasion that is activated upon host cell perforation by the major virulence factor of L. monocytogenes, the pore-forming toxin listeriolysin O (LLO). LLO is known to mediate the disruption of the endocytic vesicle that contains newly internalized bacteria and the secondary vesicle formed during cell-to-cell spreading, leading to the release of L. monocytogenes into the cytoplasm, where it proliferates. It was previously thought that LLO was only active within acidified endosomes. However, our laboratory and others have shown that LLO released by extracellular bacteria efficiently perforates host cells and plays critical roles i this context. In particular, our published data demonstrate that LLO is sufficient to induce L. monocytogenes internalization into epithelial cells. To gain insight into this novel invasion pathway, we constructed a LLO variant that binds to host cells and assembles into a prepore complex, but is unable to form a pore. This unique tool allowed us to demonstrate that host cell perforation is required for LLO-induced bacterial internalization. Our recent data also show that in cells injured by LLO, ionic fluxes across the plasma membrane are master regulators of bacterial internalization and survival pathways that maintain host cell integrity. This application will elucidate host cell responses to ionic fluxes induced by LLO and how these responses control bacterial internalization and host cell survival. We will use state-of-the-art live-cell FRT fluorescence microcopy to identify and characterize signaling events underlying LLO-dependent bacterial internalization and cell survival. We will also test the role of LLO in bacterial internalization in vivo using the murine model. Aim 1 will elucidate signaling and endocytic pathways activated by LLO to induce F-actin-dependent L. monocytogenes internalization. Aim 2 will elucidate the mechanisms of plasma membrane repair in cells perforated by LLO and the cross-talk between membrane repair and host cell invasion in vitro and in vivo. Elucidating host cell responses to plasma membrane damage caused by pathogens and how these responses affect the course of infection are emerging areas of research. The novel invasion mechanism studied in this application is relevant to numerous pathogens as pore-forming toxins are common virulence factors of viruses as well as prokaryotic and eukaryotic intracellular parasites. This work is expected to identify host pathways that can be targeted for therapeutic interventions against a wide range of diseases caused by intracellular pathogens.